A-D. Induced firing of dopaminergic neurons dramatically decreased sleep during the dark period in light-dark or constant darkness conditions followed by a sleep rebound the following day when firing was returned to normal levels. In constant darkness, sleep was even more severely suppressed with both subjective daytime and nighttime sleep almost entirely absent. In A-D, TH-Gal4 driven expression of dTrpA1 was used to transiently increase the activity of dopaminergic neurons when the temperature was raised from 21 °C to 27 °C at the beginning of the night. The behavior was monitored for 3 days either in light-dark or constant darkness at 27 °C before returning to 21 °C. For simplicity, only one day of data from each condition is shown. The data was collected from control UAS-dTrpA1 (blue), control TH-Gal4 (green), and TH-Gal4:UAS-dTrpA1 (orange).

A-B. Membrane tethered GFP fragment CD4::spGFP1-10 was expressed in most of the dopaminergic neurons with TH-Gal4 and CD4::spGFP11 was expressed in l- and s-LNvs with pdfLexA. Green is GFP staining and red is PDF staining. A. The fine fibers in the ventral elongation are likely to be the dendrites of the l-LNvs . Reconsitituted GFP signals were detected around the LNv cell bodies and dendritic area, but not in the optical lobe around the axons of the l-LNvs (N=6). The diagram indicates the orientation of the brain. D and M indicate the dorsal and medial side of the brain, respectively. B. An image with higher magnification shows the reconstituted GFP signals around the LNv cell body and dendritic area. Note, the anti-PDF staining in the dendritic areas is very weak because the dendrites do not likely contain much of the PDF peptide, resulting in GFP that does not appear to colocalize well with anti-PDF staining in the dendritic areas. C-D. Membrane tethered GFP fragment CD4::spGFP1-10 was expressed in most of the octopaminergic neurons with Tdc2-Gal4. Reconsitituted GFP signals were also detected around the LNv cell bodies and dendritic area (N=10). Scale bar is 10 µM.

A. Example showing how FRET images were processed using an automated method as described in METHODS and MATERIALS. Briefly, each video has two channels (YFP and CFP). The responses of a cell to a drug can be computed as the mean of its CFP/YFP ratios, which are normalized by signals captured under the untreated condition. Cells without statistically significant response differences over time are merged as a group. In this example, the l-LNvs, but not the s-LNvs, increased cAMP in response to bath application of dopamine. B. Dopamine application induced stronger responses in the l-LNvs compared with octopamine. (Left, flies reared in light-dark conditions were used for imaging. Right, flies reared in constant darkness day 1 were used). C. The responses can be induced by a dopamine agonist and are blocked by a dopamine antagonist. The average fluorescence change (area under the “relative cAMP change” curve) was determined by calculating an average CFP/YFP ratio increase from 100s to 445s. Error bar represents SEM. A dopamine agonist, 100uM pergolide mesylate, also induced an increase of cAMP in the l-LNvs with an effect only slightly less than dopamine alone. The l-LNv dopamine-induced cAMP response was almost completely blocked following a 15 min pre-incubation with a dopamine antagonist, 50uM (+)-Butaclamol hydrochloride. D. dopamine-induced responses are cell-autonomous; the l-LNv response to dopamine in both the presence and absence of TTX was indistinguishable. The l-LNvs increased cAMP level in response to bath application of dopamine in light-dark conditions. Responses of individual brain samples from different times of the day are shown. The relative cAMP changes are calculated as the normalized CFP/YFP ratio. Each curve represents the average cAMP response of all the visible l-LNvs in one hemisphere. The average cAMP responses from 13 brains are shown. Colored curves, TTX was added to the acutely dissected brains before bath application of dopamine. Grey curves, responses were recorded without TTX.

A-C. Light exposure suppressed the l-LNv responses to dopamine. Flies were housed in light-dark conditions (A) or constant darkness conditions (B) and the response to dopamine during daytime or subjective day is compared with that during nighttime or subjective night. C. Summary of the relative changes of cAMP shown in A and B. The l-LNv responses to dopamine during the day/subjective day versus the night/subjective night are not significantly different within either light-dark or constant darkness conditions. However, comparison between light-dark and constant darkness conditions showed that the responses of the l-LNvs to dopamine in constant darkness are much stronger during both the subjective day and subjective night than the responses at the same circadian times in light-dark conditions. D-F. Daytime light exposure suppressed the nighttime l-LNv responses to octopamine. Flies were housed in light-dark conditions (D) or constant darkness conditions (E) and the response to octopamine during daytime or subjective day is compared with that during nighttime or subjective night. Note that the response amplitude of l-LNvs from subjective day in constant darkness was similar to that of daytime in light-dark conditions. F. Summary of the relative changes in cAMP shown in D and E. The responses to octopamine during daytime, nighttime, or subjective daytime were similar while the l-LNvs from subjective night were more sensitive to octopamine. p are significant difference from control groups (student’s t-test). Error bar represents SEM.

The l-LNv responses to dopamine were not affected by PER. The daytime (A) and nighttime responses (B) are plotted separately. The dopamine-induced responses of the l-LNvs from control brains are compared with those from per⁰¹ mutant flies. C. Summary of the relative changes of cAMP shown in A and B. The responses to dopamine were not affected by per⁰¹ mutation. D-F. PER positively regulates octopamine evoked responses by l-LNv at night. Flies were housed in light-dark conditions and the daytime (D) and nighttime responses (E) are plotted separately. The octopamine-induced responses of the l-LNvs from control brains are compared with those from per⁰¹ mutant flies. F. Summary of the relative changes of cAMP shown in D and E. The responses to octopamine during daytime were not affected by per⁰¹ mutation (left), while the nighttime responses were dramatically decreased in the per⁰¹ mutants (right).

A-B. dD2R negatively regulates dopamine evoked responses in the l-LNvs. A. The l-LNv response to dopamine in light-dark conditions was dramatically increased by knocking down dD2R expression in the l-LNvs. The dopamine-induced responses of the l-LNvs from control brains are compared with those from dD2R-RNAi knockdown mutant flies. B. The l-LNv response to dopamine in constant darkness conditions was not affected by knocking down dD2R expression in the l-LNvs. The dopamine-induced responses of the l-LNvs from control brains are compared with those from dD2R-RNAi knockdown mutant flies. C. Summary of the relative changes of cAMP shown in A and B. The responses in constant darkness are comparable with those in dD2R knockdown mutants in light-dark conditions.